The present invention relates generally to Alzheimer""s disease, and more specifically to methods and compositions for use in diagnosis and treatment of Alzheimer""s Disease.
Alzheimer""s disease (AD) is a devastating, neurodegenerative progressive disorder first recognized in 1907 (Alzheimer, Algemeine Zeitschrift fur Psychiatrie 64:146-148, 1907). AD is a common disease in the elderly and is the predominant cause of dementia in people over 65 years of age. The prevalence of AD is estimated to be as high as 18.7% and 47.2% for the 75-84 year and xe2x89xa785 year age groups respectively. Thus, there is a large affected population in most countries of the world.
Clinical symptoms of the disease typically begin with subtle short term memory problems. As the disease progresses, difficulty with memory, language, and orientation worsen to the point of interfering with the ability of the person to function independently. Other symptoms, which are variable, include myoclonus and seizures. Duration of AD from the first symptoms of memory loss until death is 10 years on average, but may range from 6-8 years to more than 20 years. AD always results in death, often from respiratory-related illness.
The pathology in AD is confined exclusively to the central nervous system (CNS). The predominant features are amyloid deposits (plaques) and neurofibrillary tangles (NFT). In AD, amyloid is found associated with the vascular system of the CNS and as focal deposits in the parenchyma. The major molecular component of amyloid is a highly hydrophobic peptide called the Axcex2 peptide. This peptide aggregates into filaments in an anti-xcex2-pleated sheet structure resulting in the birefringent nature of the AD amyloid. While Axcex2 is the major component of AD amyloid, a partial list of other proteins associated with the amyloid includes xcex1-1-anti-chymotrypsin (Abraham, et al., Cell 52:487-501, 1988), cathepsin D (Cataldo, et al., Brain Res. 513:181-192, 1990), non-amyloid component protein (Ueda, et al., Proc. Natl Acad. Sci. USA 90:11282-11286, 1993), apolipoprotein E (apoE) (Namba, et al., Brain Res. 541:163-166, 1991; Wisniewski and Frangione, Neurosci. Lett. 135:235-238, 1992; Strittmatter, et al., Proc. Nat. Acad. Sci. USA 90:1977-1981, 1993), apolioprotein J (Choi-Mura, et al., Acta Neuropathol. 83:260-264, 1992; McGeer, et al., Brain Res. 579:337-341, 1992), heat shock protein 70 (Hamos, et al., Neurology 41:345-350, 1991), complement components (McGeer and Rogers, Neurology 43:447-449, 1992), xcex1 2-macroglobin (Strauss, et al., Lab. Invest. 66:223-230, 1992), interleukin-6 (Strauss, et al., Lab. Invest. 66:223-230, 1992), proteoglycans (Snow, et al., Lab. Invest. 58:454-458, 1987), and serum amyloid P (Coria, et al., Lab. Invest. 58:454-458, 1988). Surrounding many plaques are dystrophic neurites, which are nerve endings containing abnormal filamentous structures. Plaques are often surrounded by astrocytes and activated microglial cells expressing immune-related proteins, such as the MHC class II glycoproteins HLA-DR, HLA-DP, and HLA-DQ as well as MHC class I glycoproteins, interleukin-2 (IL-2) receptors, and IL- 1. The other dominant feature of AD neuropathology is the presence of NFTs. These consist of abnormal filaments bundled together in neuronal cell bodies. xe2x80x9cGhostxe2x80x9d NFTs are also observed in AD brains, which presumably mark the location of dead neurons. Other neuropathological features include granulovacuolar changes, neuronal loss, gliosis and the variable presence of Lewy bodies.
In the AD brain, the destructive process of the disease is evident on a gross level. In the late-stage of AD, ventricular enlargement and shrinkage of the brain can be observed by magnetic resonance imaging. On autopsy, extensive gliosis and neuronal loss are observed. Thus, the amyloid plaque structures and NFTs observed at autopsy are most likely the end-points of a lengthy disease process, far removed from the initiating events of AD. Also, the cells remaining at autopsy are grossly different from those of a normal brain. Neurons, which were possibly involved in initiating events, are absent and other cell types, such as the activated microglial cells and astroctyes, have gene expression patterns not observed in the normal brain. Thus, attempts using biochemical methods to identify key proteins and genes in the initiating steps of the disease are hampered by the fact that it is not possible to actually observe these critical initiating events. Rather, biochemical dissection of the AD brain at autopsy is akin to molecular archeology, attempting to reconstruct the pathogenic pathway by comparing the normal brain to the end-stage disease brain.
Substantial evidence has suggested that inherited genetic defects are involved in AD. Numerous early-onset kindreds have been described (Bird, et al., Ann. Neurol. 23:25-31, 1988; Bird, et al., Ann. Neurol. 25:12-25, 1989; Cook, et al., Neurology 29:1402-1412, 1979; Feldman, et al., Neurology 13:811-824, 1960; Goudsmit, J. Neuro.l Sci. 49:79-, 1981; Heston and White, Behavior Genet. 8:315-331, 1978; Martin, et al., Neurology 41:62-68, 1991; Nee, et al., Arch. Neurol. 40:203-208, 1983; van Bogaeert, et al., Mschr. Psychait. Neurol. 102:249-301, 1940; Wheelan, Ann. Hum. Genet. 23:300-309, 1959). (Early-onset is defined herein as onset prior to 65 years.) Families with multiple late-onset AD cases have also been described (Bird, et al., Ann. Neurol. 25:12-25, 1989; Heston and White, Behavior Genet. 8:315-331, 1978; Pericak-Vance, et al., Exp. Neurol. 102:271-279, 1988). In addition, twin studies have documented that monozygotic twins are more concordant in their AD phenotype than dizygotic twins (Nee, et al., Neurology 37:359-363, 1987; [133]). Also, the families of concordant twins have more secondary cases of AD than families of discordant twins (Rapoport, et al., Neurology 41:1549-1553, 1991).
Genetic dissection of AD has been complicated by the complexity of the disease and the limited accuracy of its diagnosis. Because AD is common in the elderly, clustering of cases in a family may occur by chance, representing possible confounding non-allelic genetic heterogeneity, or etiologic heterogeneity with genetic and non-genetic cases co-existing in the same kindred. In addition, the clinical diagnosis of AD is confounded with other dementing diseases common in the elderly.
Despite the problems associated with resolving complex genetic diseases, 2 causative AD loci and 1 risk-modifying gene have been identified. Mutations in the amyloid precursor protein (APP) gene on chromosome 21 cause early-onset ( less than 65 years) autosomal dominant AD (Goate, et al., Nature 349:704, 1991). Mutations in a recently identified gene (AD3) on chromosome 14 also result in early-onset autosomal dominant AD (Schellenberg, et al., Science 258:668, 1992; Sherrington, et al., Nature 375:754-760, 1995). For late-onset AD, the APOE gene has been identified as a genetic modifying factor (Strittmatter, et al., Proc. Natl. Acad. Sci. USA 90:1977, 1993; Corder, et al., Science 261:921, 1993; Corder, et al., Nat. Genet. 7:180-184, 1994; Benjamin, et al., Lancet 344:473, 1994; Smith, et al., Lancet 344:473-474, 1994).
The known genetic loci for AD do not account for all cases of AD. For example, in late-onset AD approximately half of AD cases do not have the APOE xcex54 allele (Brousseau, et al., Neurology 342, 1994; Kuusisto, et al., Brit. Med J. 309:636, 1994; Tsai, et al., Am. J. Hum. Genet. 54: 643, 1994; Liddel, et al., J Med. Genet. 31:197, 1994). Also, in the Volga German (VG) kindreds (Cook, et al., Neurology 29:1402, 1979; Bird, et al., Ann. Neurol. 23:25, 1988; Bird, et al., Ann. Neurol. 25:12, 1989; Bird, Am. Hist. Soc. Germ. Russia J. 49:1991; Bird, et al., in Heterogeneity of Alzheimer""s Disease, F. Boller, et al., Eds. (Spring-Verlag, Heidelberg, 1992) pp. 118-129), as in several other families with high incidence of AD, the known AD loci have been excluded as possible causes (Schellenberg, et al., Science 258:668, 1992; Lannfelt, et al., Nat. Genet. 4:218-219, 1993; van Duijn, et al., Am. J. Hum. Genet. 55:714-727, 1994; Schellenberg, et al., Science 241:1507, 1988; Schellenberg, et al., Am. J. Hum. Genet. 48:563, 1991; Schellenberg, et al., Am. J. Hum. Genet. 49:511-517, 1991; Kamino, et al., Am. J. Hum. Genet. 51:998, 1992; Schellenberg, et al., Am J. Hum. Genet. 53:619, 1993; [13]; Schellenberg, et al., Ann. Neurol. 31:223, 1992; Yu, et al., Am. J. Hum. Genet. 54:631, 1994). Identification of new genes should add considerably to the unfolding of the genetic determinants and enable biochemical and genetic approaches to diagnosis and treatment.
The present invention provides a novel, previously unidentified locus for AD, methods and compositions for diagnosis and treatment of AD, and further provides other, related advantages.
Briefly stated, the present invention provides isolated nucleic acid molecules encoding an AD4 (also known as STM2) gene product. Within one embodiment, a representative nucleic acid molecule is provided in FIGS. 1A and B (hereinafter referred to as FIG. 1). SEQ ID NO:1 Within other embodiments, nucleic acid molecules are provided which encode a mutant AD4 gene product that increases the probability of Alzheimer""s Disease (in a statistically significant manner). One representative illustration of such a mutant is an amino acid subsitution at residue 141, wherein, for example, an isoleucine may be substituted for an asparagine.
Within other aspects of the present invention, isolated nucleic acid molecules are provided, selected from the group consisting of (a) an isolated nucleic acid molecule as set forth in FIG. 1 (SEQ ID NO:1) or complementary sequence thereof, (b) an isolated nucleic acid molecule that specifically hybridizes to the nucleic acid molecule of (a) under conditions of high stringency, and (c) an isolated nucleic acid that encodes an AD4 gene product. As utilized herein, it should be understood that a nucleic acid molecule hybridizes xe2x80x9cspecificallyxe2x80x9d to an AD4 gene (or related sequence) if it hybridizes detectably to such a sequence, but does not significantly or detectably hybridize to the AD3 gene sequence under the same conditions. Within other aspects, isolated DNA molecules are provided that contain genomic sequences, such as the sequences presented in FIGS. 13-19. (SEQ ID NOS:34-40)
Within other aspects, expression vectors are provided comprising a promoter operably linked to one of the nucleic acid molecule described above. Representative examples of suitable promoters include tissue-specific promoters, as well as promoters such as the CMV I-E promoter, SV40 early promoter and MuLV LTR. Within related aspects, viral vectors are provided that are capable of directing the expression of a nucleic acid molecule as described above. Representative examples of such viral vectors include herpes simplex viral vectors, adenoviral vectors, adenovirus-associated viral vectors and retroviral vectors. Also provided are host cells (e.g., human, dog, monkey, rat or mouse cells) which carry the above-described vectors.
Within other aspects of the present invention, isolated proteins or polypeptides are provided comprising an AD4 gene product, as well as AD4 peptides of greater than 12, 13, or 20 amino acids. Within one embodiment, a protein is provided that has the amino acid sequence set forth in FIG. 2. (SEQ ID NO:2) Within another embodiment, the protein is a mutant AD4 gene product that increases the probability of Alzheimer""s Disease. Such mutants include those with an amino acid subsitution at residue 141 (e.g., an aspargine to isoleucine substitution). Within yet a further embodiment, AD4 peptides are provided which are composed of 13 to 20 amino acids derived or selected from the N-terminal, internal, or carboxy-terminal hydrophilic regions.
Within yet another aspect of the present invention, methods of treating or preventing Alzheimer""s Disease are provided, comprising the step of administering to a patient a vector containing or expressing a nucleic acid molecule as described above, thereby reducing the likelihood or delaying the onset of Alzheimer""s Disease in the patient. Within a related aspect, methods of treating or preventing Alzheimer""s Disease are provided, comprising the step of administering to a patient a protein as described above, thereby reducing the likelihood or delaying the onset of Alzheimer""s Disease in the patient. Within yet another related aspect, methods are provided for treating or preventing Alzheimer""s Disease comprising the step of administering to a patient an antibody specific for an AD4 protein as described above, thereby reducing the likelihood or delaying the onset of Alzheimer""s Disease in the patient. Within certain embodiments, the above methods may be accomplished by in vivo administration.
Also provided by the present invention are pharmaceutical compositions comprising a nucleic acid molecule, vector, host cell, protein, or antibody as described above, along with a pharmaceutically acceptable carrier or diluent.
Within other aspects of the present invention, antibodies are provided which specifically bind to an AD4 protein, or to unique peptides derived from the N-terminal, internal, or carboxy-terminal hydrophilic regions. As utilized herein, it should be understood that an antibody is specific for an AD4 protein if it binds detectably, and with a KA of 10xe2x88x927M or less (e.g., 10xe2x88x928M, 10xe2x88x929M, etc.), but does not bind detectably (or with an affinity of greater than 10xe2x88x927M, (e.g., 106M, 105M, etc.) to the AD3 protein. Also provided are hybridomas which are capable of producing such antibodies.
Within other aspects of the present invention, nucleic acid probes are provided which are capable of specifically hybridizing (as defined above) to an AD4 gene under conditions of high stringency. Within one related aspect, such probes comprise at least a portion of the nucleotide sequence shown in FIG. 1 or its complementary sequence, the probe being capable of specifically hybridizing to a mutant AD4 gene under conditions of high stringency. Within one particularly preferred aspect, probes are provided that are capable of specifically hybridizing to a mutant AD4 gene in which amino acid residue 141 is changed from asparagine to isoleucine, under conditions of very high stringency. Within other related aspects, probe are provided which are capable of specifically hybridizing to at least a portion of the nucleic sequence shown in any of FIGS. 13-19. (SEQ ID NOS:34-40) Representative probes of the present invention are generally at least 12 nucleotide bases in length, although they may be 14, 16, 18 bases or longer. Also provided are primer pairs capable of specifically amplifying all or a portion of any of the nucleic acid molecules disclosed herein.
Within other aspects of the invention, methods are provided for diagnosing a patient having an increased likelihood of contracting Alzheimer""s Disease, comprising the steps of (a) obtaining from a patient a biological sample containing nucleic acid; (b) incubating the nucleic acid with a probe which is capable of specifically hybridizing to a mutant AD4 gene under conditions and for time sufficient to allow hybridization to occur, and (c) detecting the presence of hybridized probe, and thereby determining that said patient has an increased likelihood of contracting Alzheimer""s Disease. Within another aspect, methods are provided comprising the steps of (a) obtaining from a patient a biological sample containing nucleic acid, (b) amplifying a selected nucleic acid sequence associated with a mutant AD4 gene, and (c) detecting the presence of an amplified nucleic acid sequence, and thereby determining that the patient has an increased likelihood of contracting Alzheimer""s Disease. Within yet another aspect, methods are provided comprising the steps of (a) contacting a biological sample obtained from a patient with an antibody that specifically binds to a mutant AD4 protein, under conditions and for a time sufficient to allow binding of the antibody to the protein, and (b) detecting the presence of the bound antibody. Suitable biological samples include nucleated cells obtained from the peripheral blood, from buccal swabs, or brain tissue.
Within another aspect, peptide vaccines are provided which comprise a portion of a mutant AD4 gene product containing a mutation, in combination with a pharmaceutically acceptable carrier or diluent.
Within yet another aspect, transgenic animals are provided whose germ cells and somatic cells contain an AD4 gene which is operably linked to a promoter effective for the expression of the gene, the gene being introduced into the animal, or an ancestor of the animal, at an embryonic stage. Within one embodiment, the animal is a mouse, rat or dog. Within other embodiments, the AD4 gene is expressed from a vector as described above. Within yet another embodiment, the AD4 gene encodes a mutant AD4 gene product.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.